The human milk fat globule (HMFG) has been used extensively as a source of antigenic material for the preparation of both polyclonal and monoclonal antibodies that have found widespread use in the diagnosis (Ceriani, R. L. et al, Potential Therapeutic Implications, 235-258(1986); Ceriani, R. L. et al, Immunocytochemistry in Tumor Diagnosis, 233-263(1985)) and therapy (Ceriani, R. L. et al, Cancer Res.47:532-540(1987); Ceriani, R. L. et al, Cancer Res.48:4664-4672(1988)) of breast cancer, as well as in the study of the breast epithelial cell surface and the processing of its antigenic components (Ceriani, R. L. et al, Somat. Cell Genet.9:415-427(1983); Ceriani, R. L. et al, Proc.Natl.Acad.Sci.(USA)74:582-586(1977)).
Polyclonal antiserum was originally prepared, that after appropriate absorptions with non-breast tissue was found to identify surface antigens of human mammary epithelial cells (HME-Ags) (Ceriani, R. L. et al, Proc.Natl.Acad.Sci.(USA)74:582-586(1977)). This antiserum (anti-HME) had a high specificity for normal breast epithelial cells and breast carcinomas (Ceriani, R. L. et al, Proc.Natl.Acad.Sci.(USA)74:582-586(1977)). It identified mainly three components of the human milk fat globule which had molecular weights of 150 kDa, 70 kDa, and 46 kDa, respectively.
Monoclonal antibodies were first made against the HMFG in 1980. These antibodies were applied to identify a hitherto unknown component of the breast epithelial cell surface, a large molecular weight mucin-like glycoprotein, that was named non-penetrating glycoprotein (NPGP) (Ceriani, R. L. et al, Somat. Cell Genetics 9:415-427(1983)). This latter component appears to be extremely antigenic in the mouse. The vast majority of monoclonal antibodies prepared against HMFG as well as breast tumors have been found to have specificity against different epitopes of this mucin complex. Less frequently, monoclonal antibodies have been prepared against the 70 kDa and 46 kDa components of the HMFG (Ceriani, R. L. et al, Somat. Cell Genetics 9:415-427(1983); Peterson, J. A. et al, Hybridoma, in press (1990)).
The reason for the high immunogenicity of NPGP has recently been elucidated by the characterization of cDNA clones selected from a lambdagt11 breast cell library using both polyclonal and monoclonal antibodies against the mucin (Gendler, S. J. et al, Proc.Natl.Acad.Sci.85:2320-2323(1988); Gendler, S. et al, Biological Chemistry 263:12820-12823(1988)).
These cDNA clones consist of large arrays of highly conserved 60 bp tandem repeats (Gendler, S. J. et al, Proc.Natl.Acad.Sci.85:2320-2323(1988); Siddiqui, J. et al, Proc.Natl.Acad.Sci.85:2320-2323(1988)). The resulting 20 amino acid repeat contains epitopes for several anti-mucin antibodies (Taylor-Papadimitriou et al, Breast Cancer Immunodiagnosis and Immunotherapy Plenum Publishing Corp. New York, in press (1989); Xing, P. X. et al, Breast Cancer Immunodiagnosis and Immunotherapy, Plenum Publishing Corp. New York, in press (1989)).
The repeat is apparently unstable at the genomic level. This may account for the observed polymorphism seen at the gene, RNA and protein levels for this high molecular weight mucin (Siddiqui, J. et al, Proc.Natl.Acad.Sci.85:2320-2323(1988); Gendler, S. et al, Biological Chemistry 263:12820-12823(1988)). An initial report on cDNA cloning of the mucin product suggested that the core protein had a molecular weight of about 68 kDa (Gendler, S. J. et al, Proc.Natl.Acad.Sci.85:2320-2323(1988)). However, the mRNA was found to be large enough to code for proteins from about 170 kDa to 230 kDa (Gendler, S. J. et al, Proc.Natl.Acad.Sci.85:2320-2323(1988); Siddiqui, J. et al, Proc.Natl.Acad.85:2320-2323(1988)); Gendler, S. et al, Biological Chemistry 263:12820-12823(1988)). More recently, using milder deglycosylation methods, a core protein was identified having a molecular weight of about 200 kDa (Abe, M. et al, Cancer Res.49:2834-2839(1989)).
Attention has also been devoted to the study and use of the NPGP mucin complex, largely as a result of its high immunogenicity. Thus, a large number of monoclonal antibodies were prepared against it. However, the smaller components of HMFG also appear to be important molecules on the surface of breast epithelial cells. They have a breast specificity as demonstrated by the anti-HME antibodies (Ceriani, R. L. et al, Proc.Natl.Acad.Sci.USA74:582-586(1977)).
The 46 kDa and 70 kDa HME antigens are found in serum of breast cancer patients and thus can be used as markers for breast cancer in serum assays (Ceriani, R. L. Proc.Natl.Acad.Sci.USA79:5420-5424(1982)). In addition, the 70 kDa component has been found to co-purify with the intact mucin complex and has been reported to be associated with the NPGP mucin complex by means of disulfide bonds, making it a possible linker protein of this surface mucin complex (Duwe, A. K. et al, Biochem.Biophys.Res.Comm.165:1305-1311(1990)).
Further evidence supporting the importance of the about 70 kDalton component of the surface of breast epithelial cells comes from the work of Imam et al, (Imam A. et al, Biochem.J. 193:47-54(1981)). Imam, et al, have purified a 70 kDalton glycoprotein from HMFG membranes that they have termed epithelial membrane glycoprotein (EMGP-70). They have prepared polyclonal antibodies against it and found that the antisera identifies this component on the apical surface of luminal epithelial cells of all ducts and lobules of the mammary gland. It is also present on normal apocrine but not eccrine sweat glands coils and ducts of skin. This antisera also binds to breast carcinomas and has been used to suggest the breast epithelial origin of both mammary and extra-mammary Paget's disease (Imam A. et al, Br.J.Cancer 3:373-378(1988)).
Imam et al (1988), supra, also prepared polyclonal antibodies against a major component of the HMFG that had a molecular weight of 155 kDaltons and found that antisera bound also to the apical surface of lobules and terminal ducts, but not larger ducts of mammary gland. The latter also did not bind to apical surface of normal apocrine and eccrine sweat gland coils and ducts, or sebaceous glands in skin. The MFGM-gpl55 did become localized in Paget's disease and breast disease but not in cases of extramammary disease (Imam A. et al, Br.J.Cancer 3:373-378(1988)).
Other authors (Heid, H. W. et al, Biochem.Biophys.Acta 2:228-238(1983)) have characterized a 70 kDalton glycoprotein component from HMFG that they have termed butyrophilin. It is also present on the apical plasma membrane of lactating mammary gland of human breast (Heid, H. W. et al, Biochem.Biophys.Acta 2:228-238(1983)). Heid et al found that two predominant polypeptides in diverse species having molecular weights of 155 kDaltons and 67 kdaltons. The 155 kDalton component was identified by Heid et al as xanthine oxidase and the 67 kDalton component was termed butyrophilin (Heid, H. W. et al, Biochem.Biophys.Acta 2:228-238(1983)). The 67 kDalton molecule is said to be attached to the inner face of the membrane (Heid, H. W. et al, Biochem.Biophys.Acta 2:228-238(1983)).
However, up to the present time the sequences of the different molecular weight polypeptide components or the DNAs and RNAs encoding them are not known. Nor have antibodies been prepared to the recombinant polypeptide sequences containing the epitopes responsible for normal and cancerous breast epithelial cell specificities.